Pre-mRNA splicing takes place in the nucleus of eukaryotic cells and is mediated by the spliceosome. The major components of the spliceosome are the small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U5, U4/U6, U11, U12, and U4atac/U6atac, each of which comprises one U snRNA molecule, a common core of seven Sm proteins (B/B′, D1, D2, D3, E, F and G), and several snRNP-specific proteins (Luhrmann, 1990, Mol. Biol. Rep. 14: 183-192; Luhrmann, 1990, Biochim. Biophys. Acta 1087: 265-292; Will and R. Luhrmann, 2001, Curr. Opin. Cell Biol. 13: 290-301).
The biogenesis of snRNPs occurs in the cytoplasm shortly after the nuclear export of nascent snRNAs and requires the assembly of the Sm proteins into a seven-member ring (Kambach, 1999, Cell 96: 375-387; Stark, 2001, Nature 409: 539-542) on a consensus sequence (PuAU4-6GPu) known as the Sm site of the U snRNA (Branlant, 1982, EMBO J. 1: 1259-1265; Nagai, 2001, Biochem. Soc. Trans. 29: 15-26). After formation of the Sm core, the 7-methyl guanosine cap of the snRNA is hypermethylated to become a 2,2,7-trimethyl guanosine (TMG) cap (Mattaj, 1986, Cell 46: 905-911; Plessel, 1994, Mol. Cell. Biol. 14: 4160-4172). A properly assembled Sm core, cap hypermethylation and 3′-end processing are required for the translocation of the mature snRNPs into the nucleus, where they function in splicing (Fischer and Luhrmann, 1990, Science 249: 786-790; Fischer, 1993, EMBO J. 12: 573-583; Hamm, 1990, EMBO J. 9: 1237-1244; Mattaj, 1993, Mol. Biol. Rep. 18: 79-83; Mattaj and De Robertis, 1985, Cell 40: 111-118; Will and Luhrmann, 2001, Curr. Opin. Cell Biol. 13: 290-301).
The process of bringing the protein and RNA components together during U snRNP assembly in the cytoplasm is mediated by and dependent upon the survival of motor neurons (SMN) protein complex (Buhler, 1999, Hum. Mol. Genet. 8: 2351-2357; Fischer, 1997, Cell 90: 1023-1029; Liu and Dreyfuss, 1996, EMBO J. 15: 3555-3565; Liu, 1997, Cell 90: 1013-1021; Meister, 2001, Nat. Cell. Biol. 3: 945-949; Meister and Fischer, 2002, EMBO J. 21: 5853-5863; Pellizzoni, 1998, Cell 95: 615-624; Pellizzoni, 2002, Science 298: 1775-1779; Yong, 2004, Mol. Cell. Biol. 24: 2747-2756; Yong, 2002, EMBO J. 21: 1188-1196; Yong, 2004, Trends Cell Biol. 14: 226-232). Reduced levels of SMN due to a genetic defect cause spinal muscular atrophy (SMA), a severe neuromuscular disease that is characterized by degeneration of motor neurons in the spinal cord (Cifuentes-Diaz, 2002, Semin. Pediatr. Neurol. 9: 145-150; Crawford and Pardo, 1996, Neurobiol. Dis. 3: 97-110; Iannaccone, 2004, Curr. Neurol. Neurosci. Rep. 4: 74-80). SMN, as an oligomeric protein, is part of a large multi-protein complex that contains Gemin2 (Liu, 1997, Cell 90: 1013-1021), the DEAD box RNA helicase Gemin3 (Charroux, 1999, J. Cell Biol. 147: 1181-1194), Gemin4 (Charroux, 2000, J. Cell Biol. 148: 1177-1186), Gemin5 (Gubitz, 2002, J. Biol. Chem. 277: 5631-5636), Gemin6 (Pellizzoni, 2002, J. Biol. Chem. 277: 7540-7545), and Gemin7 (Baccon, 2002, J. Biol. Chem. 277: 31957-31962). Although the function of the SMN complex in snRNP assembly is its best characterized activity, it most likely functions in the assembly, metabolism and transport of various other RNPs, including snoRNPs, miRNPs, and the machineries that carry out transcription and pre-mRNA splicing (Buhler, 1999, Hum. Mol. Genet. 8: 2351-2357; Friesen and Dreyfuss, 2000, J. Biol. Chem. 275: 26370-26375; Gubitz, 2004, Exp. Cell Res. 296: 51-56; Jones, 2001, J. Biol. Chem. 276: 38645-38651; Meister, 2000, Hum. Mol. Genet. 9: 1977-1986; Mourelatos, 2001, EMBO J. 20: 5443-5452; Mourelatos, 2002, Genes Dev. 16: 720-728; Narayanan, 2004, Mol. Cell 16: 223-234; Pellizzoni, 2001, Curr. Biol. 11: 1079-1088; Pellizzoni, 1999, Proc. NatI. Acad. Sci. USA 96: 11167-11172; Pellizzoni, 2001, J. Cell Biol. 152: 75-85; Pellizzoni, 1998, Cell 95: 615-624).
Purified snRNP total proteins, a preparation referred to as TPs, readily assemble a Sm core on a minimal Sm sequence oligonucleotide in vitro without ATP hydrolysis or other non-snRNP factors (Raker, 1999, Mol. Cell. Biol. 19: 6554-6565; Raker, 1996, EMBO J. 15: 2256-2269; Sumpter, 1992, Mol. Biol. Rep. 16: 229-240). However, in cell extracts the biogenesis of U snRNPs requires ATP hydrolysis (Kleinschmidt, 1989, Nucleic Acids Res. 17: 4817-4828; Meister, 2001, Nat. Cell. Biol. 3: 945-949; Pellizzoni, 2002, Science 298: 1775-1779), indicating that snRNP proteins are not free to randomly associate with any uridine-rich RNA sequences in cells. Rather, it is the SMN complex that actively brings Sm proteins to U snRNAs, acting as a crucial specificity factor to ensure that highly stable Sm cores are only assembled on the correct snRNAs (Pellizzoni, 2002, Science 298: 1775-1779; Yong, 2004, Mol. Cell. Biol. 24: 2747-2756; Yong, 2004, Trends Cell Biol. 14: 226-232). Several components of the SMN complex bind directly to the Sm proteins, including the binding of SMN to the RG-rich C-terminal domains of the Sm proteins B, D1, and D3 (Baccon, 2002, J. Biol. Chem. 277: 31957-31962; Brahms, 2001, RNA. 7: 1531-1542; Buhler, 1999, Hum. Mol. Genet. 8: 2351-2357; Charroux, 1999, J. Cell Biol. 147: 1181-1194; Charroux, 2000, J. Cell Biol. 148: 1177-1186; Friesen and Dreyfuss, 2000, J. Biol. Chem. 275: 26370-26375; Gubitz, 2002, J. Biol. Chem. 277: 5631-5636; Liu, 1997, Cell 90: 1013-1021; Pellizzoni, 2002, J. Biol. Chem. 277: 7540-7545; Pellizzoni, 1999, Proc. Natl. Acad. Sci. USA 96: 11167-11172). This interaction is enhanced by the symmetric dimethylarginine (sDMA) modification of specific arginines by the 20S methylosome that contains an arginine methyltransferase (JBP1/PRMT5) (Friesen, 2001, Mol. Cell 7: 11111-1117; Friesen, 2001, Mol. Cell. Biol. 21: 8289-8300; Friesen, 2002, J. Biol. Chem. 277: 8243-8247; Meister, 2001, Curr. Biol. 11: 1990-1994). The SMN complex also binds directly and with sequence specificity to the Sm-site containing U snRNAs. For U1 snRNA, the SMN complex binding domain is contained within stem-loop 1 (SL1) (Yong, 2002, EMBO J. 21: 1188-1196). For the other major U snRNAs, U2, U4, and U5, the minimal SMN complex-binding domains are closer to their 3′-ends and contain the Sm site and the 3′ stem-loop. These SMN complex-binding domains are necessary and sufficient for SMN complex binding and SMN-dependent assembly of Sm cores (Yong, 2004, Mol. Cell. Biol. 24: 2747-2756; Yong, 2004, Trends Cell Biol. 14: 226-232). Previous studies have suggested that the SMN complex contains at least two separate high-affinity RNA binding domains—one for U1 snRNA and the other for U2, U4 and U5 snRNAs (Yong, 2004, Mol. Cell. Biol. 24: 2747-2756; Yong, 2004, Trends Cell Biol. 14: 226-232).
The herpesvirus saimiri (HVS)-encoded small nuclear RNAs (HSURs 1-7) also use the SMN complex to assemble Sm cores (Murthy, 1986, EMBO J. 5: 1625-1632; Golembe, 2005, Mol. Cell. Biol. 25: 602-611). The HSURs bind the SMN complex with very high affinity to the U4 snRNA-type binding site and can effectively out-compete host snRNAs for snRNP assembly (Golembe, 2005, Mol. Cell. Biol. 25: 602-611). The Sm sites and predicted secondary structures of the HSURs are simple, conserved and have apparently evolved to closely resemble those of U2, U4, and U5 snRNAs (Lee, 1988, Cell 54: 599-607; Lee, 1990, J. Virol. 64: 3905-3915).
There have been many different attempts to identify small, stable and specific nucleic acids for manipulating, investigating and inhibiting the transcription and splicing complexes, and therefore gene expression. As an example, both antisense nucleotides and siRNA have been employed to silence or otherwise suppress the transcription of any number of genes. However, despite much promise and some clinical success, delivery, stability, toxicity, specificity and the delivery capacity of antisense and siRNA molecules, particularly into the cell nucleus, have always hampered this technology. The present invention addresses these issues and meets the need for small, stable, specific and deliverable gene-expression modulating nucleic acids.